Herpes simplex virus (HSV) 1 and 2 are common ?-herpesviruses that establish lifelong latency. While most HSV infections of mucosal tissues are relatively benign, there are rare cases of encephalitis and HSV infections of the cornea can produce inflammatory pathology known as herpes stromal keratitis (HSK). HSK frequently involves recurring infections, often over years, produced by virus that reactivates in sensory ganglia that then travels to the cornea, producing scarring which can eventually lead to blindness. In the U.S. there are 60,000 cases of HSK/annually and HSV remains the leading infectious cause of blindness. Recurrent HSV infections in mucosa and the eye stem from reactivation of latent virus in neurons followed by anterograde transport in neuronal axons, a process by which virus particles hitchhike on motors that ferry virus from neuron cell bodies to axon tips. Anterograde transport is a fundamentally important process for ?-herpesviruses and involves at least two distinct stages. The first stage involves assembly of virus particles in the cytoplasm, followed by sorting of these virus particles in Golgi/endosomal compartments into axons. The second stage involves kinesin motors that transport virus particles along microtubules within axons. Our studies of the cytoplasmic stages of anterograde transport will focus on two HSV membrane proteins gE/gI and US9 that cooperate to promote the assembly of virus particles and the polarized sorting of particles into axons. HSV virus lacking both gE and US9 are unable to transport virus particles into the most proximal portions of axons, supporting defects in cytoplasmic events leading to axonal transport. Upon further characterization, we discovered that gE-/US9- double mutants were unable to assemble enveloped virus particles and, instead, viral capsids accumulated on cytoplasm membranes. There was also evidence that gE/gI and US9 participate in a subsequent process, involving sorting of enveloped virions into axons. These observations represent the first example of neuron-specific defects in HSV assembly and sorting and represent a new paradigm for how gE/gI and US9 function in neurons. The research in Aim 1 will test two hypotheses: i) gE/gI and US9 promote assembly by collecting other viral assembly proteins on cytoplasmic membranes that are sites of virus envelopment and ii) following envelopment, gE/gI and US9 trafficking sequences function to sort these particles into axons. The molecular mechanisms involved in gE/gI and US9 mediated assembly and sorting will be investigated using novel high resolution imaging techniques coupled with a panel of viral mutants and biochemical assays. In Aim 2, we will address another fundamentally important question: ?which of the many kinesin motors transport HSV particles in axons?? To address this question, we will take advantage of recent advances in our ability to transduce neurons using baculovirus vectors to deliver fluorescent cargo molecules, kinesins, ?split kinesins? and miRNAs to silence kinesin expression and determine the functional significance of specific kinesins.